Factor viii compositions and methods of making and using same

a technology of fviii inhibitors and compositions, applied in the field of factor viii, can solve the problems of limited treatment options for patients with fviii inhibitors, affecting product yield and quality control, heterogeneous product mixtures, etc., and achieves the effects of improving efficacy, enhancing pharmacokinetic and pharmacologic properties, and convenient dosing

Inactive Publication Date: 2020-03-19
BIOVERATIV THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides new coagulation factor VIII fusion protein compositions for the treatment of hemophilia A and other hemorrhaging disorders. These fusion proteins consist of factor VIII and an extended recombinant polypeptide called XTEN. The use of XTEN allows for improved pharmacokinetic and pharmacologic properties compared to unmodified factor VIII, resulting in more convenient dosing and better efficacy. The patent also describes a method for determining the amount of fusion protein needed for effective treatment and a pharmaceutical regimen for achieving hemostasis in hemophilia A patients. The results show that the use of factor VIII-XTEN fusion protein significantly improves parameters associated with hemophilia A disease compared to unmodified factor VIII.

Problems solved by technology

As such, treatment options for patients with FVIII inhibitors are limited.
However, the conjugation step can result in the formation of heterogeneous product mixtures that require extraction, purification and / or other further processes, all of which inevitably affect product yield and quality control.
Also, the pharmacologic function of coagulation factors may be hampered if amino acid side chains in the vicinity of its binding site become modified by the PEGylation process.
Unfortunately, the Fc domain does not fold efficiently during recombinant expression, and tends to form insoluble precipitates known as inclusion bodies.
These inclusion bodies must be solubilized and functional protein must be renatured from the misfolded aggregate, which is a time-consuming, inefficient, and expensive process.

Method used

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  • Factor viii compositions and methods of making and using same
  • Factor viii compositions and methods of making and using same
  • Factor viii compositions and methods of making and using same

Examples

Experimental program
Comparison scheme
Effect test

example 1

ion of XTEN_AD36 Motif Segments

[0387]The following example describes the construction of a collection of codon-optimized genes encoding motif sequences of 36 amino acids. As a first step, a stuffer vector pCW0359 was constructed based on a pET vector and that includes a T7 promoter. pCW0359 encodes a cellulose binding domain (CBD) and a TEV protease recognition site followed by a stuffer sequence that is flanked by BsaI, BbsI, and KpnI sites. The BsaI and BbsI sites were inserted such that they generate compatible overhangs after digestion. The stuffer sequence is followed by a truncated version of the GFP gene and a His tag. The stuffer sequence contains stop codons and thus E. coli cells carrying the stuffer plasmid pCW0359 form non-fluorescent colonies. The stuffer vector pCW0359 was digested with BsaI and KpnI to remove the stuffer segment and the resulting vector fragment was isolated by agarose gel purification. The sequences were designated XTEN_AD36, reflecting the AD family...

example 2

ion of XTEN_AE36 Segments

[0390]A codon library encoding XTEN sequences of 36 amino acid length was constructed. The XTEN sequence was designated XTEN_AE36. Its segments have the amino acid sequence [X]3 where X is a 12 mer peptide with the sequence: GSPAGSPTSTEE (SEQ ID NO: 23), GSEPATSGSETP (SEQ ID NO: 24), GTSESATPESGP (SEQ ID NO: 25), or GTSTEPSEGSAP (SEQ ID NO: 26). The insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:

AE1for:(SEQ ID NO: 1628)AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGAAE1rev:(SEQ ID NO: 1629)ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCTAE2for:(SEQ ID NO: 1630)AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCCAE2rev:(SEQ ID NO: 1631)ACCTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCTAE3for:(SEQ ID NO: 1632)AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCCAE3rev:(SEQ ID NO: 1633)ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGTAE4for:(SEQ ID NO: 1634)AGGTACYTCTACYGAACCKTCYGARGGYAGCGCWCCAE4rev:(SEQ ID NO: 1635)ACCTGGWGCGCTRCCYTCRGAMGGTTCRGTAGARGT

[0391]We also annealed the phosphoryl...

example 3

ion of XTEN_AF36 Segments

[0393]A codon library encoding sequences of 36 amino acid length was constructed. The sequences were designated XTEN_AF36. Its segments have the amino acid sequence [X]3 where X is a 12 mer peptide with the sequence: GSTSESPSGTAP (SEQ ID NO: 27), GTSTPESGSASP (SEQ ID NO: 28), GTSPSGESSTAP (SEQ ID NO: 29), or GSTSSTAESPGP (SEQ ID NO: 30). The insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs:

AF1for:(SEQ ID NO: 1636)AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCCAF1rev:(SEQ ID NO: 1637)ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGAAF2for:(SEQ ID NO: 1638)AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCCAF2rev:(SEQ ID NO: 1639)ACCTGGAGAWGCRGARCCGCTTTCMGGRGTAGARGTAF3for:(SEQ ID NO: 1640)AGGTACYTCYCCKAGCGGYGAATCTTCTACYGCWCCAF3rev:(SEQ ID NO: 1641)ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGTAF4for:(SEQ ID NO: 1642)AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCCAF4rev:(SEQ ID NO: 1643)ACCTGGRCCMGGAGATTCWGCRGTAGAGCTRGTRGA

[0394]We also annealed the phosphorylated oli...

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Abstract

The present invention relates to compositions comprising factor VIII coagulation factors linked to extended recombinant polypeptide (XTEN), isolated nucleic acids encoding the compositions and vectors and host cells containing the same, and methods of making and using such compositions in treatment of factor VIII-related diseases, disorders, and conditions.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application is a continuation of U.S. patent application Ser. No. 14 / 379,192, filed Feb. 20, 2015, which is a 35 U.S.C. ยง 371 filing of International Patent Application No. PCT / US2012 / 046326, filed Jul. 11, 2012, which claims priority to U.S. Provisional Patent Application Ser. No. 61 / 599,400, filed Feb. 15, 2012, the entire disclosures of which are hereby incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]Factor VIII is an important component of the intrinsic pathway of the blood coagulation cascade. In the circulation, factor VIII is mainly complexed to von Willebrand factor. Upon activation by thrombin, (Factor IIa), it dissociates from the complex to interact with factor IXa in the intrinsic coagulation cascade, which, in turn, activates factor X. Once removed from the von Willebrand factor complex, activated factor VIII is proteolytically inactivated by activated Protein C (APC), factor Xa, and factor IXa, and is q...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C07K14/755
CPCC07K14/755A61K38/00C07K2319/31C07K2319/00A61P7/00A61P7/02A61P7/04
InventorSCHELLENBERGER, VOLKERCHANG, PEI-YUNVARFAJ, FATBARDHADING, SHENGSILVERMAN, JOSHUAWANG, CHIA-WEISPINK, BENJAMINSTEMMER, WILLEM P.GEETHING, NATHANKULMAN, JOHNLIU, TONGYAOTOBY, GARABET G.JIANG, HAIYANPETERS, ROBERTWANG, DEPINGMEI, BAISONG
OwnerBIOVERATIV THERAPEUTICS INC